Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

Pseudouridine (?) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), where is contributes ~10% of the total uridine level. However, ? constitutes only ~0.3% of the uridines present on mRNAs and its effect on mRNA function remains unclear. ? residues have however been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of ? residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based ? mapping technique called photo-crosslinking assisted ? sequencing (PA-?-seq) and then use it to map ? residues on not only multiple cellular RNAs but also the mRNAs and genomic RNA encoded by HIV-1. We also describe several 293T-derived cell lines in which human PUS enzymes previously reported to add ? residues to mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were individually inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of ? addition on cellular mRNAs to each of these three PUS enzymes, the ? sites present on HIV-1 transcripts remained unaffected. However, loss of PUS1 function did significantly reduce HIV-1 gene expression by a presumably indirect mechanism. Overall design: Photoactivated UV crosslinking of pseudouridine antibody to pseudouridilated RNA, followed by immunoprecipitation and sequencing (PA-?-Seq). RNA from 293T or CEM cells, WT or pseudouridine synthatase (Pus) knockout cells